Synthetic genes for bovine parainfluenza virus

ABSTRACT

A method is disclosed for constructing a synthetic gene for the production of a viral protein or portion thereof. The method is useful for genes found on minus-strand RNA viral genomes, such as those of rhabdoviruses or paramyxoviruses. The protein or a portion thereof prepared by expression of the synthetic gene in a suitable host may be used for vaccine or diagnostic purposes.

BACKGROUND OF THE INVENTION

This invention relates to the production of vaccines against bovineparainfluenza virus type 3 (PI-3), which is classified as aparamyxovirus. More specifically, synthetic DNA genes coding for PI-3proteins may be produced as disclosed herein. These genes may be usedfor the production of the selected viral protein by transformed cells,the biosynthetic protein being useful in a vaccine, diagnostic kit orthe like. The synthetic gene itself will be useful as a diagnosticagent.

Bovine parainfluenza virus type III, also called PI-3 or shipping fevervirus, has considerable pathologic and economic impact as a principalfactor in the initiation of an acute respiratory disease syndrome incattle. Shipping fever syndrome can be initiated by PI-3 viralinfection, usually under stressful conditions such as those associatedwith the shipping or feedlot management of cattle. The viral infectionis believed to predispose the animals to bacterial infection, forexample by Pasteurella multocida or Pasteurella haemolytica, whichresults in shipping fever syndrome. Annual economic losses due todiminished body weight, expensive treatments and delayed marketabilityare estimated in excess of $75 million. A similar virus infects sheep,leading to a respiratory disease very similar to shipping feversyndrome.

Viral vaccines, including live attenuated vaccines, have been employedfor about 20 years with some success. For example, live bacterialvaccines produced from chemically modified strains of Pasteurellamultocida and Pasteurella haemolytica are disclosed in U.S. Pat. No.4,293,545 (Kucera).

The success of the viral vaccine approach for the prevention ofparainfluenza viruses has been restricted by the limited effectivenessof current vaccines. This is due, in part, to interference from existingantibodies or inhibition by other viral vaccines. In addition, use oflive viral vaccines on breeding animals may result in fetal infectionand subsequent abortion.

Recently, it has become possible to produce a synthetic gene byformulating a bimolecular double-stranded DNA copy of a messenger RNA(mRNA) molecule. The synthetic gene produced in this manner will codefor that protein which was the translation product of the selected mRNA.An example of this genetic engineering process is disclosed in U.S. Pat.No. 4,357,421 (Emtage et al.) where it is used to produce a syntheticgene for an influenza haemagglutinin protein. Influenza viruses containno DNA; rather, they contain a segmented negative strand viral RNA(vRNA) genome which is replicated during infection to produce viralmessenger RNA (mRNA) and templates for the production of further vRNA.Influenza virus genomes are of the segmented type, that is, each gene ispresent as a separate piece of vRNA which is transcribed separately toproduce mRNAs. The process disclosed by Emtage et al. utilizes isolatedvRNA as a direct template for the synthetic gene of interest.

SUMMARY OF THE INVENTION

It now is possible to construct a synthetic gene or DNA fragment whichcorresponds to a viral gene located on the nonsegmented, negative (orminus) strand vRNA genome of the PI-3 virus. Cells infected with thevirus are obtained and the mRNA--both viral and host RNA--is separatedfrom other cell components. Double-stranded synthetic DNA moleculescomplementary to the original mRNAs are constructed. This complementaryDNA (cDNA) population is cloned to prepare a cDNA gene library fromwhich viral specific genes may be selected. The viral gene of interestis identified and isolated by, for example, reciprocal hybridization andhybrid-selected translation. Once the gene of interest is identified, itis inserted into an appropriate vector which is used to transform asuitable host for expression of the gene. The expressed protein may beused to formulate subunit vaccines, diagnostic agents and the like.

It is an object of this invention to provide a method whereby syntheticgenes can be constructed which correspond to genes normally found on thegenomes of the bovine parainfluenza type 3 virus, a paramyxovirus.

It is a closely related object to identify and isolate the syntheticgene coding for a particular viral target protein, for example, anantigenic protein such as hemagglutinin.

It is a further object to express the selected synthetic gene intransformed hosts in order to economically manufacture the viral targetprotein coded for by the synthetic gene.

It is an additional object to provide a method for producing DNAsequences from which protein amino acid sequences can be determined inorder to design synthetic peptides.

Moreover, it is intended that diagnostic DNA prooes and peptidediagnostic agents be provided by using the process of this invention.

The following abbreviations have been used throughout in describing theinvention:

bp--base pairs

BSA--bovine serum albumin

cDNA--complementary DNA

Ci--curie

cm--centimeter

dATP--deoxy-adenosine triphosphate

dCTP--deoxy-cytidine triphosphate

dGTP--deoxy-guanosine triphosphate

dTTP--deoxy-thymidine triphosphate

DTT--dithiothreitol

DNA--deoxyribonucleic acid

EDTA--ethylenediaminetetraacetic acid

GuSCN--guanidine thiocyanate

HA--hemagglutinin

HEPES--N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid

IU--International unit

M--mole

MDBK--Madin-Darby bovine kidney (cells)

MET--methionine

μ--micro

mM--millimole

mRNA--messenger RNA

NC--nucleocapsid protein

oligo(dA)--polymer made up of a few (usually 2-20) deoxyadenosinemolecules

oligo(dT)--polymer made up of a few (usually 2-20) deoxythymidinemolecules

³² P--radioactive phosphorus, mass no. 32

%--percent

PI-3--bovine parainfluenza virus type 3

PIPES--piperazine-N,N'-bis[2-ethane sulfonic acid]

poly-A--poly-deoxyadenosine

poly-C--poly-deoxycytidine

poly-G--poly-deoxyguanosine

RNA--ribonucleic acid

rRNA--ribosomal RNA

SDS--sodium dodecylsulfate

SSC--0.15 M sodium chloride - 0.015 sodium citrate (pH 7.0)

TNE--10 mM Tris-HCl (pH 8.0) - 100 mM sodium chloride - 1.0 mM EDTA (pH8.0)

Tris-HCl--Tris(hydroxymethyl)aminomethane-HCl

tRNA--transfer RNA

U--unit

v/v--volume/volume

vRNA--viral RNA

w/v--weight/volume

X--any amino acid residue

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, in four parts, is a representation of the DNA sequence of thebovine parainfluenza virus hemagglutinin (HA) cDNA cloned by the methodof this invention, including the restriction enzyme cleavage sites.

FIG. 2 is a table listing the restriction enzyme sites in the clonedcDNA of FIG. 1.

FIG. 3 is a translation of the DNA sequence of the HA cDNA gene of FIG.1 into the corresponding amino acid sequence.

FIG. 4 is the predicted amino acid sequence of the HA protein which willbe the translation product of the HA cDNA gene of FIG. 1.

DESCRIPTION OF THE INVENTION

In this description of the invention, references to "mRNA" and "cDNA"are meant to refer to the whole RNA or DNA molecule, respectively, or toany biologically significant portion thereof. That is, it is intendedthat the method of this invention be practiced either with respect to acomplete viral mRNA molecule or with respect to a sub-portion of theviral mRNA molecule. For example, it may be desired to isolate, copy andclone only that portion of a viral gene which codes for a particularprotein subunit or portion thereof, or for a particular antigenic site.

PI-3 virus is classified as a paramyxovirus on the basis ofmorphological, serological and biochemical properties. Paramyxovirusesare "RNA viruses" and contain no DNA. Their genetic information iscontained solely in a single-stranded, continuous (or non-segmented)piece of RNA. Members of the paramyxovirus family exhibit a replicationstrategy based on a negative stranded RNA genome. The general scheme ofparamyxovirus RNA replication entails the transcription of subgenomicmRNA species from the negative-stranded template from a single promotorby a virion-associated transcriptase complex. In those systems whichhave been studied, each mRNA species is associated with polyribosomes,is polyadenylated and generally encodes a single viral protein.

The replication of PI-3 has received little study, although severalviral structural proteins have been identified. See, for example,Shibuta et al., "Characterization of Bovine Parainfluenza Virus Type 3,"Microbiol. Immunol., Vol. 23, pp. 617-28 (1979) and Guskey et al., "HighYield Growth and Purification of Human Parainfluenza Type 3 Virus andInitial Analysis of Viral Structural Proteins," J. Gen. Virology, Vol.54, pp. 115-23 (1981). The prior art discloses no information regardingthe intracellular proteins or the viral mRNA species synthesized duringviral replication. Moreover, transcription and coding assignments forthe PI-3 mRNAs are not available, except as disclosed herein.

The invention disclosed herein enables one to construct a discretesynthetic gene or DNA fragment corresponding to a gene located on thenonsegmented, minus strand vRNA genome of the PI-3 virus. The syntheticgene or fragment codes for a PI-3 viral protein or portion thereof andcomprises a double-stranded DNA gene which is a copy of the viral RNAgene coding for that protein or portion thereof. The synthetic gene maybe used for the production of the protein of interest, or "target"protein. "Target" protein will be used herein to designate both theviral protein of interest and the protein produced by the expression ofthe synthetic gene. It should be understood, however, that the expressedprotein may differ in insignificant ways from the viral protein, or maycorrespond to only a portion or subunit of the viral protein.

As a preliminary step to constructing a synthetic gene, the targetprotein is identified. Where preparation of a subunit vaccine ordiagnostic agent is desired, the target protein may be identified bydemonstrating that it can stimulate the formation of neutralizing orprotective antibodies by an infected animal. In the case of the PI-3virus, viral hemagglutinin (HA) is the principal structural proteinagainst which protective antibodies are generated. The HA protein isfound on the surface of the virus and is responsible for attachment ofthe virus to the cells for initiation of the infection process.Isolation of the gene coding for the hemagglutinin protein is desired sothat it can be cloned and used for the extra-viral production of the HAprotein. This biosynthetic HA protein is particularly suitable for usein vaccines. It will stimulate the production of antibodies against PI-3in vaccinated animals without risking viral infection because the HAprotein itself is not infectious. However, there is evidence that otherstructural proteins also may contribute to the triggering of antibodies.The activity of a vaccine or diagnostic agent can be futher enhanced bythe inclusion of biosynthetic versions of these other structuralproteins. For example, structural fusion protein, which is a viralsurface protein responsible for fusion to the cell to be infected andhaving properties which cause transfer of the viral RNA to those cells,may be desired for inclusion in a vaccine or the like.

Host cells infected with the virus are obtained by conventional means.It is preferred that Madin-Darby bovine kidney (MDBK) cells be used asthe host cells, since they support high levels of viral replication. Thecells are infected with the virus and prepared according to standardcell culture techniques.

The RNA is harvested from the cells by any convenient means. Forexample, the guanidine thiocyanate-CsCl method of Chirgwin et al.,Biochemistry, Vol. 18, pp. 5294-99 (1979), which is described in moredetail in Example I, may be used. Total RNA, that is the host mRNA, tRNAand rRNA as well as viral vRNA and mRNA, is isolated or recovered.

The mRNA of all viruses infecting eukaryotes naturally occurs inpolyadenylated (poly-A-tailed) form. It is preferred to take advantageof this in separating mRNA from other cell components and from otherRNAs. To isolate polyadenylated mRNA, the total recovered RNA mostconveniently is fractionated on oligo(dT) cellulose columns. However,other methods may be used to separate the mRNA, if desired.

From polyadenylated mRNAs, it is possible to construct double-strandedhybrid RNA/DNA molecules using the technique of reverse transcription.The enzyme reverse transcriptase, an RNA-directed DNA polymerase, willsynthesize, on each molecule, a new strand of DNA complementary to theexisting strand of RNA. This transcription requires the presence of adouble-stranded growing point or primer where transcription may begin.The primer may be constructed by hybridizing an oligo(dT) molecule tothe 3'-poly-A end of the mRNA. The reverse transcriptase beginssynthesizing cDNA at the double-stranded growing point, producing thedouble-stranded mRNA/cDNA hybrid molecule.

Next, the mRNA strand of each double-stranded molecule is digested orremoved in a manner which leaves the cDNA strand intact. For example,the RNA strand may be digested by alkaline hydrolysis or withribonuclease, or removed from the cDNA strands by heat denaturation. Theresult is single-stranded cDNA, which is a self-priming structure with ahairpin loop at its 3' end.

The single-stranded cDNA molecules are converted into double-strandedcDNA by using reverse transcriptase or another DNA polymerase, such asE. coli DNA polymerase (E.C. 2.7.7.7), or using a Klenow fragment of aDNA polymerase (formed by cleaving the polymerase molecule withtrypsin). Any of these enzymes will construct a second DNA strandcomplementary to the first single-strand cDNA molecule, withtranscription beginning at the partially double-stranded hairpin endprimer. The resulting molecule is substantially double-stranded cDNA,with a remaining single-stranded hairpin end. By treating the moleculeswith S1 nuclease (E.C. 3.1.30.1), a single-strand specific nuclease, thesingle-stranded hairpin end is trimmed. The result is a population ofdouble-stranded cDNA molecules which are complementary to the originalpopulation of mRNA molecules.

It should be remembered that the RNA molecules used as templates in thisprocess comprise both viral and host mRNAs. As a result, the cDNApopulation prepared by the method described above is quiteheterogeneous. The cDNAs are cloned to generate a library or gene bankfrom which the cDNA gene of interest can be identified.

The cDNA clone library is prepared by inserting the cDNA genes intosuitable vectors for transformation of suitable hosts. Host-vectorcombinations for this stage of the procedure should be chosen for easein manipulation and consistency of excision. In addition, the vectorshould contain a single restriction endonuclease recognition site whichpreferably is suitable for being re-formed by homopolymeric tailing ofthe cDNA and the digested vector. For example, the plasmid vectorpBR322, suitable for transforming E. coli, may be digested with Pst Irestriction endonuclease (E.C. 3.1.23.31) to produce a linear DNAmolecule. This linear molecule may be tailed using terminaldeoxynucleotidyl transferase (E.C. 2.7.7.31) and dGTP to yield poly-Gtails on the vector. Poly-C tails may be added to the cDNA moleculesusing terminal deoxynucleotidyl transferase and dCTP. The poly-G tailson the vector are complementary to the poly-C tails on the cDNAs.

The products (in this embodiment, poly-C tailed cDNAs and poly-G tailedlinear vectors) then may be mixed and annealed by heating and slowlycooling to form reconstituted plasmids having the cDNA genes inserted atthe original Pst I restriction site. Upon annealing, one strand at eachend of the inserted cDNA will have a gap corresponding to the Pst Isite. Once the recombinant plasmid is introduced into a transformationhost cell, the host will repair the gaps, reconstructing the Pst I siteat either end of the inserted gene. This construction permits thesubsequent excision of the cDNA gene by the use of Pst I restrictionendonuclease for further manipulation.

There are alternative procedures which can be used to insert the cDNAgenes into vectors. For example, it is possible to join the fragmentswith linker molecules using an appropriate DNA ligase. Alternatively,homopolymeric tailing may be used to create complementary poly-A andpoly-T sequences at the ends of each fragment.

The chimeric plasmids or vectors thus constructed are used to transformcells suitable for transformation by the vector utilized. E. coli isparticularly well suited as a transformation host, but other organisms,Bacillus subtilis, for example, may be used with appropriate vectorselection. From the cDNA clone library thus constructed, clonescontaining viral-specific genes may be identified and isolated.

A sub-clone bank comprising only viral specific clones, that is,transformed host cells which comprise viral specific gene or DNAfragments, is formed using the technique of differential hybridization.Single stranded cDNA probes are prepared from viral-infected and fromuninfected MDBK cells by the method described above, except that ³² PdCTP is used in order to obtain radiolabeled cDNA. Cloned E. coli cellsfrom the cDNA library previously constructed are grown on nitrocellulosefilters and duplicate filters are prepared. The colonies are lysed andthe released nucleic acid fixed to the filters. The radiolabeled probesare hybridized to the cloned cDNA on the filters. After unbound probe isremoved, viral specific colonies, that is, colonies comprising cDNAcorresponding to a viral gene, may be identified using autoradiography.Positive colonies which read positive only on filters hybridized usingthe cellular-plus-viral cDNA probe, and not on the duplicate filtershybridized using the cellular mRNA probe, are viral-specific cDNAclones. Only these clones need be studied in the identificationprocesses described below.

In order to facilitate the screening process, reciprocal hybridization(or colony cross-hybridization) analysis may be conducted to defineindividual groups of clones within the viral-specific cDNA clone librarywhich correspond to separate viral genes. This step is not requiredsince identification can be made by transformation and directexpression, followed by immunologic identification using, for example,monoclonal antibodies. However, since there may be large numbers ofclones in the library, it will be more efficient to streamline theprocess by creating groups of clones which cross-hybridize within thegroup and therefore are considered to contain segments of the same viralgene.

In reciprocal hybridization, a single viral-specific clone is digestedwith Pst I to release the cDNA insert. The insert then is separated,radiolabeled with ³² P, and hybridized against all the clones in thePI-3 cDNA library. All clones hybridizing with the probe are designatedGroup A. A second probe is derived from one of the nonhybridizing clonesand the procedure repeated to form Group B, etc., until multiple groupsare formed, and the clones are grouped to the extent possible ordesired. In the PI-3 virus embodiment discussed herein, Groups A and Ccontained the largest number of clones and, based on analogy to mRNAabundance patterns of other paramyxoviruses (Rozenblatt et al., "Cloningand Characterization of DNA Complementary to the Measles Virus mRNAEncloding Hemagglutinin and Matrix Protein," J. Virology, Vol. 42, pp.790-97 (1982)), these groups were presumed to encode the viralnucleocapsid protein gene and the HA protein gene, respectively, asshown in Table I.

Northern blot analysis may be used to determine the coding assignmentsof each of the clone groups, using a prototype clone from each group. Bythis technique, the clones may be correlated to the correspondingprogenitor mRNAs. Individual prototype clones are radiolabeled and usedas probes to detect viral mRNAs from infected cells which have beenblotted to nitrocellulose filters. The viral mRNAs may be givententative coding assignments based on the similarities of sizerelationships to virion proteins and to mRNAs of other related viruses,as determined, for example, by electrophoresis. Six distinct poly-A RNAspecies, designated RNA 1-6, are found in PI-3 infected but notmock-infected cells. Mock-infected cells are prepared according to thesame procedures as viral-infected cells, without the addition of theviral agent. Using vesicular stomatitis virus mRNAs, or other mRNAs ofknown size, as markers, the molecular weights of the PI-3 mRNAs may beestimated.

As shown in Table I, the coding capacity of the six PI-3 poly-A RNAspecies correlates roughly with the sizes of the six viral structuralproteins and with the estimated total coding capacity of the viralgenome. The Group A probe (mRNA 1) hybridized at a positioncorresponding to the putative nucleocapsid mRNA and the Group C probe(mRNA 3) hybridized at a position corresponding to the putative HA mRNA.No hybridization to mRNA from uninfected host cells (i.e., host mRNA)was observed.

Hybrid-selection and in vitro translation of viral mRNA may be used toconfirm coding assignments and thus definitively identify individualclones encoding the viral target protein, e.g., the HA protein. By theseprocedures, poly-A mRNAs from viral-infected and control cells can becompared for their ability to direct viral-specific protein synthesis ina mRNA-dependent cell-free translation system.

Plasmid cDNA from clones in each group are isolated and immobilized onnitrocellulose filters. Poly-A mRNAs derived from virus-infected ormock-infected (control) cells are hybridized with the filter-bound cDNA.The mRNAs which specifically hybridize to each of the immobilized cDNAsare eluted from the filters and translated in vitro. Analysis of thetranslation products by a technique such as polyacrylamide gelelectrophoresis (PAGE) may be used to confirm the identification of theclones with respect to the viral protein encoded by the cDNA of eachclone Group. It may be desired also to use immunologic analysis of thein vitro expressed proteins because proteins which are glycosylated whenproduced in vivo will not be glycosylated in vitro and therefore willnot migrate to the same positions on the gel. In the PI-3 embodiment,the Group A clones select mRNA which directs the synthesis of a proteinwhich migrates with the PI-3 nucleocapsid protein; Group C clones selectmRNA which directs synthesis of a protein migrating with the HA protein.No viral-specific proteins should be detectable in extracts of thetranslation products of mRNAs from the mock-infected control cells.

Next, the clone encoding the target protein may be characterized byrestriction enzyme analysis and/or nucleotide sequence determination.Tnis step will be of particular interest and importance if it is desiredto construct a gene encoding only a particular portion of the targetprotein, such as the portion containing an active antigenic site on theprotein. Alternatively, this information may be used to design andconstruct a synthetic protein corresponding to the active site of thetarget protein, or for the study and rational design of antiviral drugs.These characterization procedures are carried out by conventionalmethods and techniques. The FIGURES show the results of thecharacterization of the PI-3 HA gene. FIG. 1 is a representation of theDNA sequence of the gene cloned as described above. FIG. 2, which liststhe restriction enzyme sites of the gene, FIG. 3, a translation of theDNA sequence of the HA gene, and FIG. 4, the predicted amino acidsequence of the HA protein which will be formed, were generated bycomputer analysis of the DNA sequence of the gene.

Referring to FIG. 1, the DNA sequence and restriction enzyme recognitionand cleavage sites are fully detailed. By convention, the DNA sequenceis read in a 5' to 3' direction. The HA cDNA insert shown in this FIGUREis 1675 base pairs in length, including the poly-G and poly-C tails usedfor cloning. Without the tails, this cDNA clone represents 1633 basesderived from the PI-3 HA mRNA. The clone represents a substantiallycomplete cDNA copy of the HA mRNA; primer extension sequencing of the HAmRNA obtained from infected cells indicates that only about 10 to 15bases are missing at the 5' end of the gene. In the gene of FIG. 1, thefirst translation initiation codon (ATG) encoding a methionine (MET)residue is found at position 40 in the sequence. A double stop codon(TAG TAG) has been identified near the 3' end of the cloned gene(position 1486). It is therefore apparent that FIG. 1 represents anearly complete copy of the viral HA mRNA and probably all of theprotein coding region of that mRNA.

While another initiation codon may exist further upstream in the viralmRNA, and thus is not indicated in FIG. 1, that is unlikely. Mosteukaryotic viral mRNAs are characterized by the presence of a 5'non-coding stretch of bases which contain the ribosome binding site ofthe mRNA. The putative non-coding 5'-sequence of the gene shown in FIG.1 is approximately 40 bases long. In addition, as mentioned above, about10 to 15 mRNA bases are believed to be missing from the cDNA sequence.

The non-coding 3'-sequence is characterized by a canonicalpolyadenylation signal (AAATAA) at position 1492 in the DNA sequence,immediately following the double stop codon. This sequence (underlinedin FIG. 1) signals the addition of poly-A tails to mRNAs in vivo.

FIG. 2 is a listing of all possible restriction enzyme or endonucleasesites in the cloned cDNA gene of FIG. 1. The data in the list wasgenerated by computer analysis of the DNA sequence. The first columnindicates the restriction enzyme abbreviation. The second column is therecognition or cleavage site for the enzyme and the third columnprovides the location, by base number, of each enzyme in the sequence.The numbers represent the 5' side of the cleavage site unless the enzymeabbreviation is in brackets. The brackets indicate that only arecognition site for that particular enzyme is known; the enzymeactually may cleave at a different site. The letters designating therecognition or cleavage site represent the following amino acids:

A--adenine

C--cytosine

G--guanine

J--adenine or cytosine

K--guanine or thymine

L--adenine or thymine

M--cytosine or guanine

N--adenine, cytosine, guanine or thymine

P--adenine or guanine

Q--cytosine or thymine

T--thymine

FIG. 3 indicates the translation of the DNA sequence of FIG. 1 into thecorresponding amino acid sequence. By convention, the amino acidsequence is read from amino terminus to carboxyl terminus. The cDNAclone shown in FIG. 1 is characterized by one open translation readingframe from position 1 through position 1485 of the DNA sequence where adouble stop codon is found. All other reading frames are blocked by thepresence of multiple stop codons throughout the sequence.

Measured from the first methionine residue at nucleotide postion 40 (inFIG. 3) to the termination or stop codons at position 1486, thepolypeptide encoded by this cDNA gene contains a predicted 482 aminoacid residues (shown in FIG. 4) and has a predicted molecular weight of54,142 daltons. This value is less than the observed molecular weight ofHA protein separated from virions, which is as would be expected in theabsence of glycosylation. Shown in FIG. 4 is the predicted amino acidsequence of the HA polypeptide (the target protein) with the putativeglycosylation sites (Asn-X-Ser or Asn-X-Thr) underscored with a solidline.

Hydropathicity analysis has revealed several hydrophilic and hydrophobicdomains within the protein. Of importance is the hydrophobic domain atthe extreme carboxyl terminus (residues 455 to 482) of the protein whichis characteristic of membrane proteins such as viral hemaglutinins. Thisterminal hydrophobic sequence is indicated by broken underscoring inFIG. 4.

Having identified the cDNA gene for the target protein, the gene then isinserted into an appropriate expression vector. Criteria for theselection or construction of an appropriate vector for this purpose willdepend on specific process requirements (e.g., purification, yield),product stability (i.e., susceptibility to proteases, etc.), and producttoxicity or inhibition. The choice of expression vector and host will bewithin the knowledge and skill of a person working in this art.

In assessing the appropriateness of the vector for expression of thecDNA gene, a suitable means of detecting expression of the targetprotein must be employed. Various standard immunoassay methods may beused, with detection of the target protein either by colorimetry orautoradiography. Monoclonal antibodies may be prepared, either againstthe purified target protein or the virus of interest, i.e., against eachof the proteins of the virus, for use in the immunoassay.

The recombinant plasmids, formed by inserting the cDNA gene of interestinto the selected expression vector, are used to transform hosts, whichthen are cloned. It is intended that the target will accumulate proteinwhen the transformed hosts are cultured in a suitable medium undersuitable conditions. The medium and conditions will depend on thetransformation host and selection will be within the knowledge and skillof a person working in this art. The protein expression product will beencoded by the cloned synthetic gene, under the influence of theadjacent promoter region of the plasmid. The vector preferably will havebeen engineered or selected to include a strong promoter for maximalexpression of the gene of interest. The expressed protein may beidentical or substantially identical to the viral protein of interest,may represent a portion of that protein, or may be a translation fusionprotein. In this context, a "translation fusion protein" is a peptidewhich is the product of the expression of two fused genes or segmentsthereof, that is, fused translation products. A translation fusionprotein produced by the method of this invention will comprise all orpart of the viral target protein and an amino acid sequence from a genelocated adjacent to the gene of interest on the transformaton vector orhost DNA. The translation fusion protein can be expected to beimmunologically active.

The protein product may be harvested by conventional means. For example,the host cells may be centrifuged and lysed, or supernatant containingexcreted protein may be collected. The protein may be isolated orpurified from the lysate or supernatant by molecular sievechromatography, high pressure liquid chromatography, or immunoaffinitychromatography. Monoclonal antibodies specific for the target proteinmay be used in the latter purification method.

The purified polypeptide will be antigenic and may be used as a vaccine.When administered as a vaccine, in a suitable adjuvant, the biosyntheticantigen will elicit production of antibodies to the target protein inthe treated animal or human, thus offering protection against infectionby the virus. Typically, vaccines are administered by subcutaneous orintramuscular injection, intravenously, orally or by aerosol.

The cDNA itself will be useful in assays to detect the presence of thecorresponding virus in various environments, such as the blood of ananimal thought to be infected. For example, the animal's mRNA may beisolated and immobilized on a solid support. Incubation with cDNA forthe target virus or target protein will cause the labeled viral cDNAprobe to bind to the test mRNA, if viral mRNA is present. Bound cDNA canbe detected, e.g., by colorimetry, autoradiography, etc., after unboundcDNA is removed.

Moreover, it will be possible to design and construct synthetic peptidesbased on knowledge of the nucleotide sequence of a cDNA gene constructedaccording to the method of this invention. The nucleotide sequence ofthe gene can be translated into the corresponding amino acid sequence ofthe expression protein, as in FIGS. 3 and 4. The protein itself may besynthesized chemically, or a smaller peptide may be designed andsynthesized which will incorporate one or more active or antigenic sitesof the trget protein.

The Examples which follow are given for illustrative purposes only andare not meant to limit the invention described herein.

The stock solutions and culture media used in the Examples were preparedas indicated below.

First Strand cDNA Synthesis Medium

    ______________________________________                                        Tris-HCl (pH 8.3)                                                                              0.01       M                                                 Potassium chloride                                                                             60.00      mM                                                Magnesium chloride                                                                             10.00      mM                                                dCTP             0.50       mM                                                dATP             0.50       mM                                                dGTP             0.50       mM                                                dTTP             0.50       mM                                                DTT              10.00      μg                                             Oligo(dT) (12-18)                                                                              5.00       μg/ml                                          ______________________________________                                    

Second Strand cDNA Synthesis Medium

    ______________________________________                                        HEPES (pH 6.8)   0.20        mM                                               Potassium chloride                                                                             60.00       mM                                               dCTP             0.50        mM                                               dATP             0.50        mM                                               dGTP             0.50        mM                                               dTTP             0.50        mM                                               ______________________________________                                    

Denhardt's Solution

    ______________________________________                                        Ficoll ( ™) (Pharmacacia Fine                                                                   5.0 g                                                    Chemicals, Inc.)                                                              Polyvinylpyrrolidone 5.0 g                                                    BSA (Pentax Fraction V)                                                                            5.0 g                                                    H.sub.2 O            To 500 ml                                                ______________________________________                                    

Filter through a disposable Nalgene filter. Store at -20° C.

EXAMPLE I (Isolation of Host and Viral mRNAs)

Stock Virus Preparation: Madin-Darby bovine kidney (MDBK) cells obtainedfrom the American Type Culture Collection (ATCC), 12301 Parklawn Avenue,Rockville, Maryland 30852, were grown in Minimum Essential Medium(Modified) (Dulbecco's Modification) ("DMEM") (Flow Laboratories, Inc.)supplemented with 10% fetal calf serum (K.C. Biologicals). Bovineparainfluenza virus type 3 (Strain SF-4, VR-281, obtained from ATCC) wasplaque purified twice. Virus stocks prepared by infecting MDBK cellswith a virus-saline solution at a low multiplicity of infection (<0.01PFU/cell). The infected cells were grown in DMEM at 37° C. for about 3to 4 days until cytopathalogy was nearly complete, and virus-containingsupernatants harvested, aliquoted and frozen at -70° C. until use.Stocks were titrated by plaque assay on MDBK cells under a semisolidoverlay of 2.0% (w/v) methylcellulose (Dow Chemical Co.) in DMEMsupplemented with 2% fetal calf serum, 50 I.U./ml penicillin and 50μg/ml streptomycin. Visible plaques appeared in three days, at whichtime plates were stained with crystal violet and the plaques counted.

Preparation of Tritiated PI-3 mRNA: In T-150 flasks containing infectedMDBK cells, the RNA was labeled metabolically with 20 uCi per ml ³H-uridine in the presence of 1.0 μg actinomycin D per ml at 18 hourspost-infection, the peak period of viral mRNA synthesis. The actinomycinD is used to prevent transcription of host DNA to RNA while allowingtranscription of vRNA to viral mRNA, with the result that only viralmRNA becomes labelled. Tritiated PI-3 mRNAs were used as comparisons inthe Northern blot analysis and to define the sizes of the PI-3 messages.

Isolation of RNA: Total RNA was harvested from the infected MDBK cellsusing the guanidine thiocyanate-cesium chloride method of Chirgwin etal., Biochemistry, Vol. 18, pp. 5294-99 (1979). Pursuant to this method,10⁸ from five T-150 flasks were lysed in situ by the addition of 10 mlof guanidine thiocyanate (GuSCN) buffer (5.0 M GuSCN, 50.0 mM Tris-HCl(pH 7.0), 50.0 mM EDTA, 5% (v/v) B-mercaptoethanol) adjusted to 2% (w/v)with Sarkosyl-40 (TM) (Ciba-Geigy Corp.). This mixture was heated to 55°C. for 5 minutes and then briefly chilled on ice for 5 minutes. Thelysate was layered over 7.0 ml of 5.7 M CsCl in 50 mM EDTA andcentrifuged at 20° C. for 5 hours at 15,000 x g in a swinging bucketrotor (25,000 RPM). Total RNA was recovered as a pellet from the bottomof the centrifuge tube. The RNA pellet was subjected to furtherfractionation on oligo(dT) cellulose columns to isolatepoly-A-containing mRNA according to the method of Aviv and Leder, Proc.Natl. Acad. Sci. USA, Vol. 69, pp. 1408-12 (1972).

RNA was isolated from mock-infected MDBK cells in the same manner exceptthat the cells are "infected" with saline, rather than a virus-salinesolution. RNA isolated from mock-infected cells was used for comparativepurposes in Examples IV and V.

EXAMPLE II (Synthesis of cDNA Molecules)

DNA complementary to the poly-A mRNA isolated in Example I wassynthesized by incubating 5.0 μg poly-A mRNA in 100 μl First Strand cDNASynthesis Medium at 37° C. for 10 minutes. Next, 12 U avianmyeloblastosis virus reverse transcriptase (Life Sciences, Inc.) per μgmRNA was added. This mixture was incubated at 42° C. for 45 minutes,chilled on ice for 5 minutes and heated to 100° C. for 3 minutes. It wascentrifuged at 15,000 x g in a microfuge for 2 minutes to removeprecipitated protein.

Double-stranded cDNA was synthesized from single-stranded cDNA in a 200μl incubation containing 100 μl first strand reaction supernatant in 100μl Second Strand cDNA Synthesis Medium. A volume of E. coli DNApolymerase (Klenow fragment) was added to obtain a concentration of 10 Uper μg of single-stranded cDNA. This mixture was incubated for 16 hoursat 15° C. The reaction was stopped by performing a singlephenol-chloroform extraction.

The products (double-stranded cDNA molecules) were desalted andconcentrated by two sequential spermine precipitations followed byethanol precipitation. Double-stranded cDNA in the second-strandreaction solution was precipitated in the presence of 10 mM spermine at0° C. (on ice) for 30 minutes. The precipitated cDNA was collected bycentrifugation at 15,000 x g in a microfuge for 5.0 minutes. The pelletwas resuspended in a solution containing 10 mM spermine. After a 30minute incubation at 0° C., the precipitate was collected bycentrifugation as before. Precipitated cDNA was resuspended in 400 mMsodium acetate and 10 mM magnesium acetate before being precipitatedwith 2.5 volumes ethanol at -70° C.

The double-stranded cDNA products were digested with S1 nuclease toremove the single-stranded hairpin end. The cDNA was incubated in a 50μl volume of 0.3 M NaCl, 0.03 M sodium acetate, 0.003 M ZnCl₂ (pH 4.5),and 10 U S1 nuclease for 30 minutes at 37° C. The double-stranded cDNAreaction products were desalted and concentrated by spermineprecipitation.

EXAMPLE III (Construction of cDNA Library)

The population of cDNAs prepared from mRNA from viral-infected cells inExample II was tailed by incubating with 30 U terminal deoxynucleotidyltransferase, in a reaction buffer containing 250 mM potassium cacodylate(pH 7.2), 2.0 mM CoCl₂, 1.0 mM DTT and 1.0 mM dCTP for 15 minutes at 25°C. to produce cDNA molecules with poly-C tails. The plasmid vectorpBR322 was digested with Pst I restriction endonuclease at 32° C. for 16hours to produce linear DNA molecules. These linear molecules werepoly-G tailed using dGTP by the same prodecure used to tail the cDNAs.The poly-G-tailed vector molecules were mixed with the poly-C-tailedcDNAs and annealed by incubating as follows: 70° C. for 30 minutes, 37°C. for 150 minutes and 22° C. for 30 minutes, in order to formreconstituted plasmids having the cDNA genes inserted at the originalPst I restriction site. This construction permits subsequent excision ofthe cDNA genes by Pst I. The chimeric plasmids created in this Examplewere used to transform E. coli K-12 strain AC80 (donated by L. Bopp,General Electric) cells by the calcium phosphate coprecipitation methoddescribed by Kushner, Proc. of the Int'l Symposium of Gen. Eng.,Biomedical Press (1978). The transformed E. coli cells constituted alibrary of cells containing the cDNA inserts.

EXAMPLE IV (Identification of Viral-Specific Clones)

Clones comprising viral-specific genes were identified by differentialcolony hybridizations using ³² P-labeled cDNA probes derived by reversetranscription of mRNA which was extracted by the procedure of Example Ifrom either PI-3 virus infected or mock-infected MDBK cells. Synthesisof the radiolabeled single-stranded cDNA probes was conducted by themethod described in Example II except that ³² P dCTP was substituted fordCTP in the reaction mixture.

The cloned E. coli cells prepared in Example III were grown onnitrocellulose filters overlaid onto Luria agar plates. Duplicates ofeach filter were prepared. The resulting colonies were lysed using 0.5 MNaOH, and the released nucleic acid was fixed to the filters by heatingfor 60 minutes at 80° C. in a vacuum oven. The filters containing thelysed E. coli colonies were prehybridized by incubating in 5x Denhardt'sReagent (that is, at five times the concentration given above), 5x SSC,100 μg/ml denatured salmon sperm, 50% formamide and DNA for 24 hours at42° C.

Hybridization of the radiolabeled probes to the cloned cDNA wasconducted by incubating the filters in 2x Denhardt's Reagent, 5x SSC, 50μg/ml salmon sperm DNA, 50% formamide, 7.5% dextran sulfate, andradiolabeled probe specific for either MDBK mRNA or for MDBK-plus-PI-3mRNA. Incubation was for 24 hours at 42° C. Unbound probe was removed bywashing the filters in 2x SSC plus 0.1% SDS, followed by washing in 0.1xSSC with 0.1% SDS. Viral-specific clones were detected usingautoradiography. Colonies hybridizing differentially with probescontaining viral sequences, that is, positive only on filters hybridizedusing the MDBK-plus-PI-3 mRNA probes, and negative on the duplicatefilters hybridized using the MDBK mRNA probes, were selected andrescreened to confirm viral specificity.

EXAMPLE V (Reciprocal Hybridization)

The viral-specific cDNA clones of Example IV were grouped by thereciprocal hybridization method of Rozenblatt et al., "Cloning andCharacterization of DNA Complementary to the Measles Virus mRNA EncodingHemagglutinin and Matrix Protein," J. Virology, Vol. 42, pp. 790-97(1982). Cloned DNA was excised from the pBR322 vehicle of one of theviral-specific cDNA clones by digesting with Pst I restriction enzyme.The restricted DNA was resolved by fractionation in 1.5% agarose gel,yielding linear pBR322 plasmid DNA and insert cDNA. The insert cDNA waselectroeluted from the gel and radiolabeled with ³² P-dCTP by thenick-translation procedure of Maniatis et al., Proc. Natl. Acad. Sci,U.S.A., Vol. 72, pp. 1184-88 (1975), using The Nick Translation ReagentKit available from Bethesda Research Laboratories.

The radiolabeled cDNA probe was used as a hybridization probe againstall the viral-specific clones in the PI-3 cDNA library in the followingmanner: The colonies were simaltaneously consolidated onto a master agarplate and a nitrocellulose filter on a second agar plate and allowed togrow. The colonies on the filter were lysed with alkali by the method ofHanahan and Meselson, Gene, Vol. 10, pp. 63-67 (1980). Afterneutralization, the DNA was fixed onto the filter by baking. Thesecolony DNA blots were hybridized with the ³² P-probe as described byGrunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A., Vol. 72, pp.3961-65 (1975). Following hybridization, the filters wereautoradiographed. Those clones hybridizing with the labeled probe weredesignated PI-3 Group A.

A second probe was prepared in the same manner from the nonhybridizinggroup. The hybridization procedure was repeated until all the clones hadbeen subdivided into six classes based upon the reciprocal hybridizationanalysis. Table I indicates the number of clones in each group, theapproximate size of the cDNA insert, approximate size of correspondingmRNA, molecular weight of mRNA and the tentative coding assignment,where known.

                  TABLE I                                                         ______________________________________                                        (Classification of PI-3 cDNA Clones Based Upon                                Reciprocal Hybridization Relationships)                                       Group Number:                                                                             A       B       C     D     E                                     ______________________________________                                        Probe.sup.1 6-8     5-2     3-5   5-4   6-6                                   Number of   38      8       10    3     5                                     Clones                                                                        Size Range  500-    400-    500-  400-  500-                                  of Inserts  1500bp  1300bp  1600bp                                                                              1300bp                                                                              1000bp                                Corresponding                                                                             RNA 5   RNA 4   RNA 3 RNA 6 ?                                     mRNA                                                                          mRNA size   1600    1900    1950  1190  ?                                     (bases)                                                                       mRNA molecular                                                                            0.55    0.64    0.65  0.41  ?                                     weight                                                                        (× 10.sup.6 daltons)                                                    Coding      NC      F(?)    HA    M     P(?)                                  Assignment                                                                    ______________________________________                                         .sup.1 Ten PI3 specific clones could not be classified by the reciprocal      hybridization analysis. (NC, nucleocapsid; F, fusion; HA, hemagglutinin;      M, matrix; P, phosphoprotein.)                                           

EXAMPLE VI (Northern Blot Analysis)

To determine the corresponding mRNAs for of each of the cDNA clonegroups established by reciprocal hybridization, Northern blot analysiswas carried out. The cDNA inserts from individual clones in Groups A-Dwere excised and radiolabeled with ³² P by nick-translation by themethod described in Example V for use as cDNA probes. Messenger RNA fromPI-3 virus-infected and mock-infected MDBK cells were denatured using14% glyoxal and 50% dimethyl sulfoxide in 5% sodium phosphate (pH 6.8)at 50° C. for one hour, and fractionated by electrophoresis in 1%agarose gels. The mRNAs were blot transferred to a nitrocellulose filterby capillary action, using 20x SSC.

After transfer, the filter was baked for 2 hours at 65° C., cooled toroom temperature and placed in 5x SSC for 15 minutes. It was then sealedin a bag containing prehybridization buffer (5x Denhardt's Reagent, 5xSSC, 50% formamide, and 100 μg/ml denatured (single strand) salmon spermDNA) and incubated in a 42° C. water bath for 4 hours. The filter thenwas cut into strips, each comprising a lane of PI-3 mRNA and a lane ofMDBK mRNA. The strips were placed into individual bags containinghybridization bufter (2x Denhardt's, 5x SSC, 50% formamide, 7.5% dextransulfate, 50 μg/ml denatured salmon sperm DNA and 1.0 mM sodiumpyrophosphate). Each strip was hybridized with prepared ³² P-labeledprobes, which had been denatured by heating in a boiling water bath for5 minutes prior to adding to the bag. Hybridization was carried out byincubating in a 42° C. water bath for 20 hours. Following hybridization,the filters were washed with 1.0x SSC and 0.1% SDS (4 washes, 15 minuteseach) and then with 0.5x SSC and 0.1% SDS (4 washes, 15 minutes each) atroom temperature. Next, they were autoradiographed Tritiated PI-3 mRNA,prepared in Example I, was electrophoresed in a parallel lane blotted tothe same nitrocellulose filter and visualized by fluorography usingspray EN³ Hance (TM) (New England Nuclear) to serve as an internalmarker and control. Fluorography was done by exposure to Kodak XAR (TM)film with intensifying screens at -70° C. for 24 to 48 hours, untilimages were visible.

Individual viral mRNAs were given tentative coding assignments based onthe similarities of size relationships to virion proteins and to mRNAsof other paramyxoviruses. In this Example, vesicular stomatitis virusmRNAs were used as markers in order to estimate the molecular weights ofthe PI-3 mRNAs (Rose, J. et al., "Nucleotide Sequence Complexities,Molecular Weights and Poly (A) Content of Vesicular Stomatitis VirusmRNA Species," J. of Virology, Vol. 21, pp. 1105-12 (1975). PI-3 RNAs 4and 5 migrated very closely and were best resolved when the gels wereblot transferred to nitrocellulose rather than dried beforefluorography. As shown in Table I, the coding capacity of the six poly-ARNA species correlates roughly with the sizes of the six viralstructural proteins and with the estimated total coding capacity of theviral genome. The Group A probe hybridized at a position correspondingto the putative nucleocapsid mRNA, while the Group C probe hybridized ata position corresponding to the putative HA mRNA.

EXAMPLE VII (Hybrid Selection and Translation)

These tentative coding assignments for Groups A and C were confirmed byhybrid selection and in vitro translation of viral mRNA. Plasmid DNA (10μg) was isolated from PI-3 clones of Groups A and C and purified byCsCl-ethidium bromide centrifugation. The DNA was denatured by heatingin 20 μl Tris-HCl (pH 7.5) and 1.0 mM EDTA for 10 minutes to 100° C. andquickly cooling on ice. An equal volume of 1N NaOH was added and the DNAsolution incubated at 25° C. for 20 minutes. The solution wasneutralized by the addition of 9.0 ml 1.5 M NaCl, 0.15 M sodium citrateand 0.25 M Tris-HCl (pH 8.0). The DNA was immobilized on 2.0 cm diameternitrocellulose filters (presoaked in 3x SSC) by slow filtration using avacuum manifold. The filters were air-dried for one hour at 25° C. andbaked at 80° C. for two hours in a vacuum oven.

After drying, the filters were cut into 0.7 cm strips and placed in asiliconized 30 ml corex tube. A prehybridization solution of 50% (w/v)deionized formamide, 20 mM PIPES (pH 6.4), 0.75 M NaCl, 1.0 mM EDTA, 1%(w/v) SDS, 5.0 μg single-stranded salmon sperm DNA and 5.0 μg of E. colitransfer RNA was added. This was incubated for two hours at 37° C. Theprehybridization buffer was removed and the strips washed withprehybridization buffer without tRNA and ssDNA.

For hybridizaton, the cut filters were incubated with 50 μg whole cellRNA isolated from viral-infected or mock-infected MDBK cells in ahybridization buffer of 100 μl of 50% (v/v) deionized formamide, 20 mMPIPES (pH 6.4), 0.75 M NaCl, 1.0 mM EDTA, 1% (w/v) SDS and 1.0 mMvanadyl complexes for 5 hours at 50° C. The filters were thoroughlywashed with TNE plus 0.5% SDS, and then TNE alone. The filters weretransferred to 15 ml corex tubes and 300 μl sterile water added. Elutionof the bound mRNA was conducted by boiling in a water bath for oneminute. The sample was quick-frozen in liquid nitrogen, and thawed toroom temperature. Filters were discarded. The eluted RNA was extractedwith phenol, chloroform, isoamyl alcohol and precipitated using ethanolat -70° C. The ethanol precipitated mRNA was collected by centrifugationand lyophilized.

Each eluted hybrid-selected mRNA was translated in cell-free Wheat GermExtract IVT System (Bethesda Research Laboratories) using ³⁵S-methionine (New England Nuclear). Viral-specific translation (protein)products were immunoprecipitated by using polyclonal rabbit antiseraagainst purified PI-3 virus. Immune complexes were separated fromreaction mixtures by adsorption to protein-A polyacrylamide (Immunobeads(TM), Bio-Rad) as described by Rose, Nature, Vol. 279, p.260 (1979).Immunoprecipitated products were released from the protein-A adsorbentby boiling for 5 minutes in 25 μl buffer (5% w/v SDS, 6% v/v2-mercaptoethanol in water) and electrophoresed in a 10%SDS-polyacrylamide gel system (SDS-PAGE) (Laemmli, Nature, Vol. 227 pp.680-85 (1970)). After electrophoresis, the gels were electroblotted tonitrocellulose paper (Bio-Rad), fluorographed with EN³ Hance (TM) spray(New England Nuclear), and autoradiographed for 24 hours to 1 week,until images were visible, at -70° C. using Kodak X-Omat AR (TM) film(Kodak).

As a control, viral mRNA was isolated from bovine PI-3 viral infectedcells as described in Example I. The total isolated mRNA was translatedaccording to the in vitro translation procedures described previously inthis Example. The translation products (both viral and host) wereimmunoprecipitated using polyclonal rabbit antisera against purifiedPI-3 virus as described above to identify viral specific proteins. Theproteins identified in this manner are indicated in Table II (ControlPolypeptides)

It can be seen that three viral proteins were identified by in vitrotranslation of the total mRNA, followed by immunoprecipitation. Two ofthese, the hemagglutinin (HA) and nucleocapsid (NC) proteins, correspondwell with viral specific proteins translated by the hybrid-selectedmRNAs. That is, the Group A probe hybrid-selected mRNAs which directedthe synthesis of a protein with a molecular weight of 65-70,000 daltons(corresponding to 68,000 daltons for the virion nucleocapsid protein and65-67,000 daltons for the control group protein). The Group C probehybrid-selected mRNAs which directed the synthesis of a protein with amolecular weight of 60,000 daltons (corresponding to 69,000 daltons forthe virion hemagglutinin protein and 60,000 daltons for the controlgroup protein). A 31,000 dalton protein was identified in the controlgroup which corresponds with the 35,000 dalton virion matrix protein. Noviral-specific polypeptides were detected in wheat germ extractsprogrammed with RNA from mock-infected cells.

                  TABLE II                                                        ______________________________________                                        (Molecular Weights.sup.1 of Bovine PI-3 Virus Polypeptides)                   Polypeptide                                                                           Virion       Control     Hybrid-Selected                              Designation                                                                           Polypeptides Polypeptides                                                                              Polypeptides                                 ______________________________________                                        L       180,000      ND.sup.2    ND                                           P       79,000       ND          ND                                           HA.sup.3                                                                              69,000       60,000      60,000                                       NC.sup.4                                                                              68,000       65-67,000   65-70,000                                    F       55,000       ND          ND                                           M.sup.4 35,000       31,000      ND                                           ______________________________________                                         .sup.1 Molecular weights are expressed in daltons.                            .sup.2 ND = "not detected"-                                                   .sup.3 The difference in molecular weights between the virion HA              polypeptides and the in vitro translated HA polypeptides is due to the        lack of glycosylation in the in vitro products.                               .sup.4 Nucleocapsid (NC) and matrix (M) proteins are nonglycosylated and      thus exhibit less viariability in molecular weight as determined by           SDSPAGE gels.                                                            

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, sincethese are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art withoutdeparting from the spirit of the invention.

What is claimed is:
 1. A synthetic gene or DNA fragment characterized inthat it:(a) codes for a bovine parainfluenza type-3 viral protein, and(b) comprises a double-stranded DNA gene which is a copy of the viralRNA gene coding for said protein.
 2. The synthetic gene of claim 1 whichcodes for bovine parainfluenza type-3 hemagglutinin.
 3. The DNA sequenceof the synthetic gene of claim 2 which begins with an initiation codon,ends with a termination of stop codon, and comprises the nucleotidesequence of FIG.
 1. 4. The DNA sequence of the synthetic gene of claim 2which codes for a polypeptide comprising the amino acid sequence of FIG.4.
 5. The synthetic gene of claim 1 which codes for bovine parainfluenzatype-3 structural fusion protein.
 6. The plasmid pBR322 comprising thedouble-stranded DNA gene of claim
 1. 7. The host cell E. coli comprisingthe double-stranded DNA gene of claim
 1. 8. A synthetic gene for abovine parainfluenza viral target protein, which protein is coded for inthe natural state by a gene located on the non-segmented minus strandviral genome of the bovine parainfluenza type-3 virus, produced by:(a)isolating a population of mRNA comprising the genes coding for saidviral protein, (b) constructing double-stranded mRNA/cDNA hybrids fromsaid population of mRNA using the enzyme reverse transcriptase andoligodeoxynucleotide primer molecules, (c) digesting or removing themRNA strands of said hybrids, (d) producing substantially completelydouble-stranded cDNA molecules from the single-stranded cDNA remainingafter step (c), using a DNA polymerase, and (e) trimming single-strandedend portions of said substantially completely double-stranded cDNAmolecules using a single-strand specific nuclease.
 9. The synthetic geneof claim 8 which comprises the nucleotide sequence of FIG.
 1. 10. Thesynthetic gene of claim 8 which codes for a polypeptide comprising theamino acid sequence of FIG.
 4. 11. The plasmid pBR322 comprising thesynthetic gene of claim
 8. 12. The hose cell E. coli transformed withthe plasmid of claim 11 and capable of expressing the protein of thesynthetic gene contained therein.